IMM Report Number 12

Nanomedicine: Is Diamond Biocompatible With Living Cells?

By Robert A. Freitas Jr., IMM Research Fellow

In October 1999, IMM Research Fellow Robert A. Freitas Jr. published Volume I of Nanomedicine, his ground-breaking technical work on the medical applications of molecular nanotechnology. Beginning with this issue of the Foresight Update, he will be contributing reports based on his research toward the completion of Volumes II & III of Nanomedicine.

The exteriors of many medical nanorobots [1] and nanorobot aggregates may be made of diamond. That’s why the biocompatibility of diamond surfaces and diamond particles is of considerable interest in nanomedicine [2]. Some nanomedical applications will demand a nonadhesive interface, while other applications may require complete tissue integration with the nanodevice, using biocompatible surfaces of engineered bioactivity, probably including nanostructured materials able to promote and stabilize cell attachment. Atomically-precise diamond surfaces aren’t readily available, so cell responses cannot yet be seriously investigated. However, the biocompatibility of comparatively rough, bulk manufactured diamond surfaces has been addressed experimentally by a handful of researchers  for example, in connection with diamond-coated orthopedic prostheses already proposed, developed, or in clinical use [3]. Here, I focus on one very narrow but important aspect of diamond biocompatibility: the response of living cells to diamond surfaces and particles.

Diamond Surfaces

The first pioneering study of cell response to diamond surfaces was completed by Thomson and colleagues [4] in 1991, using tissue culture plates with diamond-like carbon (DLC) coatings 0.4 microns thick. DLC is an amorphous hydrocarbon polymer with carbon bonding largely of the diamond type instead of the usual graphitic bonding [5], and thus has many of the useful properties of diamond [6]. Mouse fibroblasts grown on the DLC coatings for 7 days showed no significant release of lactate dehydrogenase (an enzyme that catalyzes lactate oxidation, often released into the blood when tissue is damaged) compared to control cells. This demonstrated that there was no loss of cell integrity due to the DLC coatings.

Mouse peritoneal macrophages similarly cultured on DLC also showed no significant excess release of lactate dehydrogenase or of the lysosomal enzyme beta N-acetyl-D-glucosaminidase (an enzyme known to be released from macrophages during inflammation). Morphological examination revealed no physical damage to either fibroblasts or macrophages, confirming the biochemical indication that there was no toxicity and that no inflammatory reaction was elicited in vitro. Follow-up studies [7] in 1994-95 found that mouse macrophages, human fibroblasts, and human osteoblast-like cells grown on DLC coatings on a variety of substrates exhibited normal cellular growth and morphology, with no in vitro cytotoxicity.

Subsequent experiments have largely confirmed these early results. For instance, human hematopoietic myeloblastic ML-1 cells and human embryo kidney cells were observed to proliferate continuously on DLC film with very high viability and no toxicity [8]. Scanning electron microscopy used to investigate the morphological behavior of osteoblasts found that these cells attached, spread and proliferated normally without apparent impairment of cell physiology when placed on DLC or amorphous carbon nitride films, whereas cells placed on silicon were able to attach but not to spread [9]. Human osteogenic sarcoma T385 cells and 1BR3 fibroblasts cultured on DLC-coated wells also showed DLC to be biocompatible [10].

The cytotoxicity study of DLC coatings by Parker and colleagues [11], employing the Kenacid Blue cytotoxicity test in vitro with 3T3-L1 mouse fibroblasts, found normal cell growth on diamond surfaces. Other tests by this team [12] of the biocompatibility of “amorphous carbon hydrogen” using a standard cell line showed that such films are nontoxic to cells, appear to increase cell attachment, and afford normal cell growth rates.

Dion et al. [13] looked at general cytotoxicity and hemocompatibility of DLC surface with 3T3 Balb/c cloned cells, and human endothelial cells isolated from placenta were investigated as a model for differentiating cells. No negative effects due to DLC coatings were observed on the viability of cells, which showed normal metabolic activities.

O’Leary and colleagues [14] evaluated cytotoxicity and cell adhesion of mouse fibroblasts on saddle field source deposited DLC (containing less than 1% hydrogen) coating a titanium alloy surface. They found normal cell adhesion, density, and spreading on DLC.

Finally, an experiment by Tang et al. [16] studied the attachment of neutrophils to plasma-preincubated ~1 cm2 350-micron-thick CVD diamond wafers. Incubation for 1 hour with purified human neutrophils at 2 x 106 cells/cm3 produced an attachment rate of ~400,000 cells/cm2 (~0.004 cells/micron2), about the same as for 316 stainless steel and 40% lower than for titanium, two common and well-tolerated implant materials. SEM photographs of CVD diamond wafers implanted intraperitoneally in live mice for 1 week revealed minimal inflammatory response.

Interestingly, on the rougher “polished” surface having ~1 micron features, a small number of spread and fused macrophages 10-13 microns in diameter were present, indicating that some activation had occurred. However, on the smoother “unpolished” surface having <0.25 micron features, samples were partially covered by round, non-spread (non-activated) cells, 4-7 microns in diameter, which had formed no obvious pseudopodia or cell bridges. The authors [16] noted that “the morphology of unpolished surfaces of CVD diamond could be responsible for preventing the activation of surface-adherent cells [but] the mechanism for this differential response of phagocytic cells…is not yet understood.” If surface rugosity [17], topography [18], or crystalline structure [19] can account for the differential response, then it is quite possible that atomically-precise diamondoid surfaces with <1 nm features  constituting much of the external surfaces of medical nanorobots and nano-organs  could be made almost completely macrophage-inactive.

Diamond Particles

In biomaterials research, it has been found [20] that even though a bulk material may be well-tolerated by the body, finely divided particles of the same material can often lead to severe and even carcinogenic complications in test animals. Differences in particle size influence histological reaction [21] and cytokine production [22]. Many nanomedical applications will involve “particle” sized diamondoid objects (e.g., micron-scale individual medical nanorobots), so it is of great interest to review the experimental data relating to the reactions of specific cells to the presence of diamond particles. We already know that finely divided carbon particles are well-tolerated by the body  the passive nature of carbon in tissue has been known since ancient times, and both charcoal and lampblack (roughly spherical 10-20 nm particles) were used for ornamental and official tattoos. Are diamond particles also well-tolerated by cells?

(1) Neutrophils. A 1982 report of possible crystal-induced neutrophil activation by 2-8 micron amorphous diamond crystals [23] has never been confirmed. Indeed, to the contrary, diamond particles are traditionally regarded as biologically inert and noninflammatory [24]. For example, Hedenborg and Klockars [25] used 4-8 micron diamond dust as an inert control in their experimental work, and found that diamond dust did not stimulate the production of reactive oxygen metabolite by polymorphonuclear (PMN) leukocytes  a proposed pathway for chronic inflammation and tissue injury of the lung. Tse and Phelps [24] found that 3-micron diamond dust crystals in a 2 mg/cm3 concentration (~0.06% Nct or “nanocrit” [2]; volume concentration) were phagocytized by 21% of all PMN cells present at a 7250 cell/mm3 concentration after 45 minutes, but no chemotactic activity was generated. Higson and Jones [26] exposed horse and pig neutrophils to urate, hydroxyapatite, pyrophosphate and brushite crystals (all implicated in joint inflammation), which induced superoxide and peroxide generation in a concentration- and temperature dependent fashion. But exposing the neutrophils to diamond crystals at 37 degrees C produced no effect. Yet another experiment [27] tested the ability of various crystals to stimulate phagocytosis, degranulation, and secretion of cell movement (motility) factors (CMFs) from PMN leukocytes and found that hydroxyapatite (HA) crystals stimulated some enzyme release and CMF generation, and monosodium urate monohydrate (MSUM) crystals much more so. But 4-8 micron diamond crystal fragments in suspension up to ~0.2% Nct, while clearly interacting with PMN leukocytes, did not stimulate degranulation, CMF production, or cell death even at high crystal concentrations.

(2) Monocytes and Macrophages. It has long been known that free carbon and diamond particles are ingested by macrophages without harmful effects. For example, cells that have taken up large amounts of 2-4 micron diamond dust remain healthy for at least 30 hours, whereas cells succumb rapidly after ingesting silica [28]. Phosphatase enzyme discharged into diamond-containing phagosomes by adherent lysosomes did not escape into the cytoplasm or nucleus. In a more recent study [29], 2-15 micron particles of diamond, silicon carbide (SiC), hydroxyapatite (HA) and polymethylmethacrylate (PMMA) were suspended in serum-free cultures of human monocytes at a concentration of 0.5 mg/cm3 (~0.01% Nct). All particles were phagocytosed. But while monocyte morphology changed after the ingestion of SiC and HA, there was no change after the ingestion of diamond, indicating no activation of the monocytes by the diamond. Interleukin-1beta production was indistinguishable for control and diamond cultures, but increased 30-fold in the HA cultures, 38-fold in the cultures exposed to SiC, and in a similar range to HA and SiC for the PMMA. The authors [29] concluded that diamond particles in serum-free monocyte culture are inert, despite being phagocytosed, unlike most other particles.

(3) Fibroblasts. Early studies in the 1950s [30] and 1960s [28] found that micron-size diamond dust particles did not induce a fibrogenic reaction. Schmidt et al. [31] note that diamond dust is nonfibrogenic in human monocyte-macrophages found in the lungs; in other words, fibroblasts are not recruited by macrophages in response to the presence of diamond dust. Diamond dust of sizes <0.5 micron and 1-2 microns did not induce the release of thymocyte proliferation factor or fibroblast proliferation factor at diamond particle concentrations up to ~0.1 mg/cm3 (~0.003% Nct) [31]. In another experiment [32], synthetic hydroxyapatite crystals at a concentration of 50 micrograms/cm3 in 1% and 10% serum stimulated 3H thymidine uptake into quiescent canine synovial fibroblasts and human foreskin fibroblast cultures. Calcium pyrophosphate dihydrate crystals also stimulated uptake, as did calcium urate crystals markedly and sodium urate crystals more modestly  but 1-5 micron diamond crystals had no mitogenic effect on the fibroblasts at particle concentrations up to 0.4 mg/cm3 (~0.01% Nct).

(4) Other Cells. The reactions of regenerating rabbit bone tissue to phagocytosable particles were studied [33] by dispersing various particles in hyaluronan and then introducing them into an implant-traversing canal, forming a bone harvest chamber. Tissue that entered the canal during the following 3 weeks was harvested. Particles of high density polyethylene, bone cement and chromium-cobalt injected in this fashion all provoked an inflammatory reaction in tissue entering the canal and caused a marked decrease in the amount of ingrown bone. But the phagocytosable 2-15 micron round-shaped diamond particles  introduced at a number density of ~60 million/cm3 (~0.7% Nct)  produced no decrease in bone formation and appeared “comparatively harmless…there was no obvious cellular reaction to these particles.” Histologically, the diamond particles aggregated into clumps. Occasional macrophages were seen nearby, but phagocytic cells remained few and dispersed, despite containing large amounts of ingested particulate diamond. There was no concentration of macrophages and giant cells such as is usually seen when PMMA or high-density polyethylene particles are implanted. Finally, diamond has never been shown to be neurotoxic [34].

(5) Inflammation and Hemolysis. Tse and Phelps [24] found that 3-micron diamond crystals in a 10 mg/cm3 concentration (~0.3% Nct) injected into canine knee joints produced “little evidence of inflammation”  intra-articular pressure remained low, along with the local cell count. A study by Dion et al. [35] observed no detectable hemolysis in vitro by various ceramic powders tested, including diamond, graphite and alumina, after 60 minutes of exposure to a powder concentration of ~0.5 gm per cm3 of diluted blood (~14% Nct).

Thus it appears that diamond is extremely  indeed outstandingly  biocompatible with living cells.

Acknowledgments

The author thanks Stephen S. Flitman, M.D., and Ronald G. Landes, M.D., for helpful comments on an earlier version of this paper.

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